Development of simple sequence repeat (ssr) core primer group based on whole genome sequence of pomegranate and application thereof

ABSTRACT

The development of a simple sequence repeat (SSR) core primer group based on the whole genome sequence of pomegranate and the applications thereof are disclosed. The primer group includes 11 primer pairs: PG080, PG130, PG139, PG152, PG153, PG140, PG098, PG070, PG077, PG090, and PG093. The SSR core primer group of the present disclosure has the advantages such as high polymorphism, good repeatability, stable amplification, and clear and easy to score bands, and is applicable to the fields of pomegranate variety identification, DNA fingerprinting construction, genetic diversity assessment and phylogenetic study, and the like, providing a new tool for pomegranate molecular marker-assisted selection and having an excellent application prospect.

RELATED APPLICATIONS

This nonprovisional application claims priority under 35 U.S.C. § 119(a) on Patent Application No. 201910631857.6 filed in China on Jul. 12, 2019, the entire contents of which are hereby incorporated by reference.

SEQUENCE LISTING

The Sequence Listing associated with the present application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference in its entirety into the specification. The name of the text file containing the Sequence Listing is entitled “US82076-SequenceListingAmendment_ST25.txt”. The text file was created on Oct. 21, 2020, having a size of about 5,000 bytes, and is being submitted electronically via EFS-Web.

FIELD

The present disclosure relates to a field of molecular biotechnology, in particular to development of a simple sequence repeat (SSR) core primer group using whole genome sequence of pomegranate and applications in pomegranate variety identification, DNA fingerprinting construction, and genetic diversity assessment and phylogenetic study.

BACKGROUND

Microsatellite (Simple Sequence Repeat, SSR) markers are short tandemly repeated motifs of 1˜6 nucleotides that abundantly present throughout the genome. SSRs have the characteristics of wide distribution, codominant inheritance, high polymorphism, convenient detection, and good stability, and are widely used in many research fields such as variety identification, genetic diversity assessment and phylogenetic relationship study, and genetic map construction and quantitative trait locus (QTL) mapping. At the early stage of SSR development, the study of SSR markers was mainly based on genomic DNA libraries or specific microsatellite-enriched libraries. Such development methods create heavy workloads, while only a very limited number of SSR markers could be obtained. In recent years, the development of SSR markers based on the whole genome sequence is able to acquire abundant markers, which could cover the entire genome and evenly distribute across the genome, so it is widely used in a variety of sequenced plants.

Pomegranate was one of the edible fruit trees recognized by humans previously, native to the Persian region (present-day Iran), and domesticated by humans in the fifth century BC. Now, pomegranate is widely cultivated in countries with Mediterranean-like climates around the world, including Tunisia, Turkey, Spain, Egypt, Morocco, the USA, China, India, Argentina, Israel, and South Africa. It is widely consumed in the form of fruits, juice, wines, and medicines due to its nutritional, medicinal, and ornamental values. According to the records, pomegranate was introduced to China when Qian Zhang served as an imperial envoy to the Western Regions, Xiyu, during the Han Dynasty, and has been cultivated in China for more than 2000 years. Pomegranate is diversified, as recorded in the “China Fruit Tree Record, Volume of Pomegranate”, there are more than 300 pomegranate genetic resources in China. Pomegranate can be divided into sweet pomegranate and sour pomegranate according to the flavor, hard-seeded pomegranate and soft-seeded pomegranate according to the hardness of seed, ornamental pomegranate and edible pomegranate according to the utilization, red skin, white skin, yellow skin, and pink skin pomegranate according to the color of pericarp, etc. Genetic exchange and variety introduction happen frequently in different local areas, and they are carried out usually basing on the local production and fruit characterization, so homonym and synonym are existed generally. The cultivation range of pomegranate is expanded gradually, which results in serious mixing between pomegranate varieties, bringing new challenges to pomegranate producers and breeders.

Based on long history of pomegranate cultivation, abundant pomegranate resources, and frequent exchanges of varieties, it is important and of practical significance to develop pomegranate variety identification, DNA fingerprinting construction, genetic diversity assessment and phylogenetic relationship study. The insensitivity of SSR markers to environmental changes and desirable genetic attributes make them valuable for variety identification and evaluating germplasm diversity. However, the SSRs of pomegranate were mainly developed from enriched genomic libraries, which is a time-consuming and laborious process. Identification of SSRs from the genome sequence has been proved to be a robust, rapid, and widely strategy. Thus, it is of great value to develop SSR marker core primers on the whole genome according to the existing genome sequence of pomegranate.

DESCRIPTION

The present disclosure is to provide rapid development of simple sequence repeat (SSR) markers using whole genome data to screen primer groups, these primers have the advantages of stable amplification, clear bands, and high polymorphism, and can be effectively applied to the fields of pomegranate variety identification, DNA fingerprinting construction, genetic diversity assessment and phylogenetic study, and the like.

According to a first aspect of the present disclosure, a developed SSR core primer group based on the whole genome sequence of pomegranate is provided. The primer group comprises 11 primer pairs including PG080, PG130, PG139, PG152, PG153, PG140, PG098, PG070, PG077, PG090, and PG093, the nucleotide sequence of each primer is sequentially shown as in Table 2 below. Each of the 11 primer pairs includes a forward primer and a corresponding reverse primer.

According to a second aspect of the present disclosure, a method for development of the SSR core primer group based on the whole genome sequence of pomegranate is provided, the method includes:

(1) pomegranate genomic deoxyribonucleic acid (DNA) extraction: a hexadecyltrimethylammonium bromide (CTAB) method is utilized to extract DNA, the extracted DNA is added with 50 μL of 0.1M Tris-EDTA (TE) buffer for dissolving overnight and then stored at −20° C. until use;

(2) whole genome data of pomegranate is downloaded from DDBJ/ENA/GenBank databases under an accession number MTKT00000000; MISA software (MIcroSAtellite identification tool) is used to mine SSR loci with different repeat units within the range of the whole genome, the SSR search criteria are 11 repeat units for dinucleotide repeats, 8 repeat units for trinucleotide repeats, 6 repeat units for tetranucleotide repeats, 5 repeat units for pentanucleotide repeats, and 4 repeat units for hexanucleotide repeats;

(3) SSR primer design

from the obtained SSR loci above, 5 SSR loci are randomly selected on each chromosome, the primers are designed by Primer 3.0 using the flanking sequences of SSRs, the parameters for the primer design are as follows: a length of the PCR products is in a range of 100˜350 bp; a melting temperature (Tm) is between 50˜70° C., ensuing a difference in Tm value between two primers does not exceed 4° C.; a GC % content is between 40˜65%; a length of the primers is in a range of 18˜28 bp; in order to ensure the specificity of the primers, the conserved flanking sequences and the SSR loci used for the primer design are at least 20˜23 bases apart; 45 primer pairs are successfully designed using the above-described method; and

(4) primer screening

genomic DNA of 6 representative pomegranate accessions from different production areas of China is amplified using the newly designed 45 primer pairs, PCR amplification is conducted in 20 μL of reaction mixture containing 1.0 ng of DNA, 0.4 μM of forward primers, 0.4 μM of reverse primers, 4 mM of MgCl₂, 400 μM of dNTPs, 1.0 U of Taq-DNA polymerase, and ddH₂O to the total volume of 20 μL. Touchdown PCR is carried out under the following conditions: 5 min at 95° C., followed by 11 cycles with a decrease of 0.8° C. in the melting temperature after each cycle {30 s at 95° C.; 30 s at 65° C.; 50 s at 72° C.}, followed by 22 cycles {30 s at 95° C.; 30 s at 55° C.; 50 s at 72° C.}, and a final extension of 8 min at 72° C.; fragment sizes of the PCR products are determined by capillary electrophoresis, 11 primer pairs with stable amplification, clear bands, and high polymorphism are selected according to the results from the amplification.

According to a third aspect of the present disclosure, an application of the developed SSR core primer group based on the whole genome sequence of pomegranate in pomegranate variety identification is provided.

The above-mentioned variety identification is to use 11 primer pairs are carried out a capillary electrophoresis with fluorescence detection. According to the results of the capillary electrophoresis detection, variety identification is determined by the number of differential loci, two varieties having differential loci ≥3 are considered as different varieties, those having differential loci <3 are considered as substantially similar or the same variety.

According to a fourth aspect of the present disclosure, an application of the developed SSR core primer group in pomegranate DNA fingerprinting construction is provided.

According to a fifth aspect of the present disclosure, genetic diversity assessment and phylogenetic research on pomegranate genetic diversity assessment and phylogenetic research applications are provided.

The beneficial effects and/or advantages of the present disclosure include:

1. The present disclosure newly develops a group of pomegranate SSR core primers, which enriched pomegranate SSR marker library.

2. The present disclosure establishes a method for developing an SSR core primer group in the pomegranate whole-genome scale. While comparing to other methods, the method of the present disclosure has the characteristics such as easy development, low cost, and high efficiency, and is able to acquire abundant markers in genome wide, which randomly distributes in 9 chromosomes, and has important practical values.

3. The 11 SSR core primer pairs developed from the present disclosure have the advantages such as high polymorphism, good repeatability, stable amplification, and clear and easy to score bands, and are applicable to the fields of pomegranate variety identification, DNA fingerprinting construction, genetic diversity assessment and phylogenetic study, and the like, providing a new tool for pomegranate molecular assisted breeding and having an excellent application prospect.

The drawing is a phylogenetic tree of 23 pomegranate accessions.

The present disclosure is further explained in combination with the implementations and drawings. The following implementations are used in the present disclosure for illustration purposes only, and are not intended to limit the scope of the present disclosure.

I. Pomegranate Genomic DNA Extraction

(1) Selection of 23 Pomegranate Accessions from Different Production Areas.

Names and origins of the 23 pomegranate accessions are shown in Table 1.

TABLE 1 Information of 23 pomegranate accessions Accession Origin AH10 Anhui, China AH14 Anhui, China AH15 Anhui, China AHHB04 Anhui, China AHHB08 Anhui, China AHHB60 Anhui, China AHHB68 Anhui, China SD35 Shandong, China SD30 Shandong, China SD41 Shandong, China SD42 Shandong, China SD47 Shandong, China SD37 Shandong, China HN5 Henan, China HN06 Henan, China HN4 Henan, China SXXA18 Shanxi, China SXXA1 Shanxi, China CY01 Xizang, Tibet Autonomous Region XJ02 Xinjiang Uygur Autonomous Region WG02 USA MK02 Xizang, Tibet Autonomous Region HY24 Anhui, China

(2) Genomic DNA Extraction Using CTAB Method.

0.2˜0.3 g of fresh leaves are weighed and added with liquid nitrogen to quickly grind into a fine powder. The powder is transferred into a 2.0 mL centrifuge tube, mixed with 1.0 mL of pre-heated (65° C.) 3×CTAB extraction buffer, and incubated into a 65° C. water bath for 1 h. After incubation, the sample is centrifuged at a speed of 12000 rpm for 10 min at room temperature, and the supernatant is transferred into a clean 2.0 mL centrifuge tube. The supernatant is added with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1, V/V/V) and gently mixed by inversion to form an emulsion. The emulsion is centrifuged at the speed of 12000 r/min for 8 min, and the supernatant is collected and added with an equal volume of chloroform/isoamyl alcohol (24:1, V/V), after gently mixing, the sample is centrifuged at the speed of 12000 r/min for 8 min. The supernatant is collected, added with an equal volume of ice-cold isopropyl alcohol and 10 μL of 3M sodium acetate, and placed for 30 min at −20° C. to precipitate. The sample is then centrifuged at the speed of 12000 r/min for 8 min, the supernatant is carefully decanted away while DNA is remained in the centrifuge tube. The DNA is washed with 75% ethanol twice and absolute ethanol once, centrifuged to remove the absolute ethanol, and allowed to dry at room temperature. The DNA is then added with 50 μL of TE buffer (0.1M) to dissolve overnight and stored at −20° C. until use.

II. Development of SSR Primers of Pomegranate Genome

(1) Whole genome data of pomegranate is downloaded from DDBRENA/GenBank databases under an accession number MTKT00000000. MISA software (MIcroSAtellite identification tool, http://pgrc.ipk-gatersleben.de/misa) is used to mine SSR loci with different repeat units within the range of the whole genome. The SSR search criteria are 11 repeat units for dinucleotide repeats, 8 repeat units for trinucleotide repeats, 6 repeat units for tetranucleotide repeats, 5 repeat units for pentanucleotide repeats, and 4 repeat units for hexanucleotide repeats.

(2) SSR Primer Design

From the obtained SSR loci above, 5 SSR loci are randomly selected on each chromosome, the primers are designed by Primer 3.0 using the flanking sequences of SSRs. The parameters for the primer design are as follows: a length of the PCR products is in a range of 100˜350 bp; a melting temperature (Tm) is between 50˜70° C., ensuing a difference in Tm value between two primers does not exceed 4° C.; a GC % content is between 40˜65%; a length of the primers is in a range of 18˜28 bp; and the primers are best to have a 5′ end of G/C and avoid a 3′ end of A. In order to ensure the specificity of the primers, the conserved flanking sequences and the SSR loci used for the primer design are at least 20˜23 bases apart. 45 primer pairs are successfully designed using the above-described method, and the primers are synthesized by Sangon Biotech Company (Shanghai, China).

(3) Primer Screening

Genomic DNA of 6 representative pomegranate accessions (AHHB04, SXXA1, CY01, XJ02, HN4, and SD47, which were originated from Anhui province, Shanxi province, Tibet Autonomous Region, Xinjiang Uygur Autonomous Region, Henan province, and Shandong province in China, respectively) is amplified using the newly designed 45 primer pairs, 11 primer pairs (Table 2) with stable amplification, clear bands, and high polymorphism are selected according to the results from the amplification.

TABLE 2 Primer sequences of 11 primer pairs Primer Forward primer Reverse primer PG080 ctgactgttgcaga aggaggtgaaacaa gagtaggctg cgaatagctg PG130 ctcatatggcgatt aagttcgataaatt ctctgtcctt gcactggtgg PG139 gtttccttccctca agtgggattttacc acccaaaa aagtcgaaca PG152 catcagaatcgtcc cagagagaagaaga ccttgtg gagaccgagc PG153 gtgtttgatgctcc gccttcaacggtct catttcattt ttcttcttct PG140 caagaaagtgtgtg ccaaaccttacccc agcgattgat tctctctctc PG098 tgccttcttaagga ctaacctcatgcac cttcaccaac ttgtcatcca PG070 cacctctgcttcag caactcaacacaat caaacaaata atccaaccca PG077 gtcagtctcctcct agacgaagcacctg tcttcaatgg agaaggaat PG090 attcttttatacta atgtcatgagagga accaaaatttgcga cccacaaa PG093 cgtcaataggacgt gatgacgtggcaga ccctgagata gtaagagagc

(4) PCR Amplification and Capillary Electrophoresis of 23 Pomegranate Accessions Using the 11 Primer Pairs

PCR amplification is conducted in 20 μL of reaction mixture containing 1.0 ng of DNA, 0.4 μM of forward primers, 0.4 μM of reverse primers, 4 mM of MgCl₂, 400 μM of dNTPs, 1.0 U of Taq-DNA polymerase, and ddH₂O to the total volume of 20 μL. Touchdown PCR is carried out under the following conditions: 5 min at 95° C.; followed by 11 cycles, with a decrease of 0.8° C. in the melting temperature after each cycle {30 s at 95° C.; 30 s at 65° C.; 50 s at 72° C.}; followed by 22 cycles {30 s at 95° C.; 30 s at 55° C.; 50 s at 72° C.}; and a final extension of 8 min at 72° C.

Fragment sizes of the PCR products are determined by capillary electrophoresis. The capillary electrophoresis is carried out by the following operations: the PCR products labeled with 6-FAM or HEX fluorescent dye are diluted 30 times using ultrapure water, 1 μL of the diluted PCR products is transferred to a deep well plate dedicated to DNA analyzer. Each well of the well plate is respectively added with 0.1 μL of GeneScan LIZ500 internal size standard and 8.9 μL of deionized formamide to denature for 5 min at 95° C. and then cool for 10 min at 4° C. After short run centrifugation of 10 s, an automatic fluorescence detection is performed by a DNA analyzer (ABI3730XL).

(5) Results and Analysis

The DNA fragments are scored on the basis of allele size. The homozygous allelic variation is recorded as X/X, where X represents the size of allelic variation at the locus. The heterozygous allelic variation is recorded as X/Y, where X and Y are two different allelic variations at the locus, small fragments in the front and large fragments in the back. The constructed fingerprinting of 23 pomegranate accessions is shown in Table 3.

TABLE 3 Fingerprint data of 23 pomegranate accessions Sample PG098 PG070 PG130 PG090 PG152 PG153 PG139 PG077 PG093 PG140 PG080 AH10 213/229 224/224 158/158 222/226 142/154 157/157 158/162 153/159 164/170 154/154 157/183 AH14 213/229 224/224 158/158 222/226 154/154 157/157 162/162 153/153 164/166 154/154 157/183 AH15 229/229 224/224 158/158 222/222 154/154 157/157 158/162 153/161 158/166 154/156 157/157 AHHB04 213/213 224/224 158/158 222/222 142/154 157/157 158/162 153/159 164/170 154/154 157/157 AHHB08 229/229 224/224 158/158 222/226 142/142 163/163 162/162 159/159 164/170 154/154 157/183 AHHB60 213/229 224/224 158/158 226/226 142/142 163/163 158/158 153/153 164/164 154/154 157/183 AHHB68 229/229 202/224 158/158 222/226 154/154 157/157 162/162 153/161 156/170 154/154 157/157 SD35 213/229 224/224 158/158 222/222 142/154 163/163 158/162 153/153 170/170 154/154 157/157 SD30 229/229 224/224 158/158 222/226 142/154 163/163 158/158 157/159 164/170 154/154 157/183 SD41 229/229 224/224 158/158 222/226 142/142 157/163 158/162 153/153 164/166 154/154 183/183 SD42 213/229 224/224 158/158 222/226 154/154 157/157 160/162 137/153 158/164 154/156 157/183 SD47 229/229 202/224 158/158 222/226 142/154 157/157 158/158 153/153 158/158 154/154 157/157 SD37 229/229 202/224 158/158 226/226 142/154 157/163 158/162 137/159 156/164 154/156 153/157 HN5 229/231 224/224 158/158 222/226 142/154 157/157 158/160 153/159 166/166 154/156 157/157 HN06 227/229 202/202 150/158 226/226 142/154 157/157 154/158 137/137 156/164 154/154 157/157 HN4 217/217 202/224 150/158 222/226 142/154 157/157 154/158 153/161 164/164 154/154 189/189 SXXA18 231/231 224/224 158/158 222/222 142/142 157/157 160/160 159/161 166/166 154/156 161/183 SXXA1 215/229 222/224 158/158 226/226 142/142 157/157 154/154 137/161 164/164 154/154 183/183 CY01 229/229 224/224 158/158 224/224 142/142 157/157 154/154 137/159 160/160 148/148 140/140 XJ02 233/233 202/202 150/150 226/226 142/154 157/157 154/154 137/159 164/164 154/154 157/157 WG02 227/229 224/224 148/156 226/226 142/154 157/157 158/158 137/159 162/164 156/156 153/153 MK02 213/213 202/224 158/158 224/224 142/142 157/157 154/154 159/159 158/158 154/154 153/153 HY24 229/229 202/202 158/158 222/222 154/154 157/157 158/162 153/153 158/170 154/154 157/157

Variety identification is determined by the number of differential loci, two varieties having differential loci >3 are considered as different varieties, those having differential loci <3 are considered as substantially similar or the same varieties. Comparing to the fingerprint data of 23 pomegranate materials, it is found that the number of differential loci between any two of the materials is greater than 3, indicating that the 11 core primer pairs may effectively identify these pomegranate resources. NTSYS-pc V2.10e software is used to calculate the coefficients of genetic similarity among varieties, an UPGMA method is used to conduct a cluster analysis to generate a phylogenetic tree (shown in the drawing). 

What is claimed is:
 1. A Simple Sequence Repeat (SSR) core primer group based on a whole genome sequence of pomegranate, the SSR core primer group comprising: 11 primer pairs comprising PG080, PG130, PG139, PG152, PG153, PG140, PG098, PG070, PG077, PG090, and PG093; wherein nucleotide sequences of each of the 11 primer pairs are sequentially shown as follows: Primer Forward primer Reverse primer PG080 ctgactgttgcaga aggaggtgaaacaa gagtaggctg cgaatagctg PG130 ctcatatggcgatt aagttcgataaatt ctctgtcctt gcactggtgg PG139 gtttccttccctca agtgggattttacc acccaaaa aagtcgaaca PG152 catcagaatcgtcc cagagagaagaaga ccttgtg gagaccgagc PG153 gtgtttgatgctcc gccttcaacggtct catttcattt ttcttcttct PG140 caagaaagtgtgtg ccaaaccttacccc agcgattgat tctctctctc PG098 tgccttcttaagga ctaacctcatgcac cttcaccaac ttgtcatcca PG070 cacctctgcttcag caactcaacacaat caaacaaata atccaaccca PG077 gtcagtctcctcct agacgaagcacctg tcttcaatgg agaaggaat PG090 attcttttatacta atgtcatgagagga accaaaatttgcga cccacaaa PG093 cgtcaataggacgt gatgacgtggcaga ccctgagata gtaagagagc


2. The SSR core primer group of claim 1, wherein the 11 primer pairs are used for pomegranate variety identification.
 3. The SSR core primer group of claim 2, wherein: the 11 primer pairs are carried out a capillary electrophoresis with fluorescence detection, according to results of amplification loci detection by the capillary electrophoresis; when a number of differential loci between two varieties is greater than or equal to 3, the two varieties are determined to be different varieties; and when the number of differential loci between the two varieties is less than 3, the two varieties are determined to be substantially similar or the same variety.
 4. The SSR core primer group of claim 1, wherein the 11 primer pairs are used for pomegranate DNA fingerprinting construction.
 5. The SSR core primer group of claim 1, wherein the 11 primer pairs are used for pomegranate genetic diversity assessment and phylogenetic study.
 6. A method for developing a Simple Sequence Repeat (SSR) core primer group based on a whole genome sequence of pomegranate, the method comprising: (1) extracting genomic deoxyribonucleic acid (DNA) of pomegranate, wherein a hexadecyltrimethylammonium bromide (CTAB) method is utilized to extract DNA, the extracted DNA is added with 50 μL of 0.1M Tris-EDTA (TE) buffer for dissolving overnight and then stored at −20° C. until use; (2) determining SSR loci with different repeat units within a range of the whole genome, wherein the SSR search criteria are 11 repeat units for dinucleotide repeats, 8 repeat units for trinucleotide repeats, 6 repeat units for tetranucleotide repeats, 5 repeat units for pentanucleotide repeats, and 4 repeat units for hexanucleotide repeats; (3) designing 45 SSR primer pairs, wherein from the obtained SSR loci, 5 SSR loci are randomly selected on each chromosome, the SSR primers are designed by Primer 3.0 using flanking sequences of SSRs, parameters for designing the SSR primers include: a length of polymerase chain reaction (PCR) products is in a range of 100-350 bp; a melting temperature (Tm) is between 50-70° C., ensuing a difference in Tm value between two SSR primers does not exceed 4° C.; a guanine-cytosine (GC) % content is between 40-65%; a length of the SSR primers is in a range of 18-28 bp; wherein the conserved flanking sequences and the SSR loci used for the SSR primer design are at least 20-23 bases apart to ensure the specificity of the SSR primers; (4) screening the 45 SSR primer pairs to select 11 primer pairs with stable amplification, clear bands, and high polymorphism, wherein: PCR amplification is conducted in 20 μL of reaction mixture containing 1.0 ng of DNA, 0.4 μM of forward primers, 0.4 μM of reverse primers, 4 mM of MgCl₂, 400 μM of dNTPs, 1.0 U of Taq-DNA polymerase, and ddH₂O to the total volume of 20 μL; touchdown PCR is carried out under the following conditions: 5 minutes at 95° C.; 11 cycles, with a decrease of 0.8° C. in the melting temperature after each cycle {30 s at 95° C.; 30 s at 65° C.; 50 s at 72° C.}; 22 cycles {30 s at 95° C.; 30 s at 55° C.; 50 s at 72° C.}; and a final extension of 8 minutes at 72° C.; fragment sizes of the PCR products are determined by capillary electrophoresis. 